Session Type: ACR Poster Session C
Session Time: 9:00AM-11:00AM
MicroRNA (miRNA) are critical gene regulators that frequently play central roles in disease. Here, we report the miRNA expression signatures of systemic sclerosis (SSc) skin and dermal fibroblasts and examined the role of Xq26.3 miRNA cluster in the exaggerated fibrotic process observed in SSc.
The global miRNA profiles of skin (10 early SSc samples and matched controls) were determined using a highly sensitive, comprehensive multiplex qPCR platform (Exiqon LNA-enhanced qPCR array) that interrogates 752 miRNAs. Similarly, global miRNA expression was investigated for early passage dermal fibroblasts from 5 SSc patients and unaffected controls. SSc patients had disease duration < 5 years and were not treated with immunosuppressive agents. Based on these studies, we then performed targeted qPCR for 3 miRNAs located in a cluster on X-chromosome in 5 SSc and control fibroblast cell lines unstimulated and stimulated with TGF-b and 8 other key Th1/Th2/Th17 cytokines for 24 hours. Additionally, we performed global gene expression microarrays of the 5 unstimulated/stimulated control fibroblasts.
Global miRNA profile of SSc whole skin samples revealed 26 dysregulated miRNAs, 3 of which were part of a miRNA cluster located in Xq26.3. The global miRNA profiling of dermal fibroblasts revealed 24 differentially expressed miRNAs in SSc. Similar to skin studies, Xq26.3 cluster miRNA were also significantly increased in SSc dermal fibroblasts. Notably, 3 of these miRNA (miR 424-5p, miR 424-3p and miR 503-5p) are predicted to target SMAD7 per in silico algorithms, therefore we investigated them further.
The fibroblast stimulation experiments in SSc and control fibroblast cell lines revealed that these 3 miRNA were significantly upregulated after stimulation by TGF-b (compared to paired unstimulated cells). Furthermore, the highest levels of these miRNA were observed after TGF-b stimulation compared to 8 cytokines (IFNa, IFNg, IL10, IL13, IL17a, IL22, MCPI, TNFa). A similar stimulation pattern was observed in SSc and control cell lines.
As predicted in silico, Xq26.3 miRNA levels were inversely correlated with SMAD7 mRNA levels (as miRNA levels increased SMAD7 levels decreased) (mir424-5p spearman r=-1 p=<0.0001, miR424-3p r=-0.9 p=0.03, miR503-5p r=-0.9 p=-0.03). No significant correlations were observed with other gene transcripts such as SMAD4 or after stimulation with other cytokines. Furthermore, there were no significant correlations with a control miRNA (miR25).
This is the first comprehensive and unbiased examination of miRNAs in SSc skin and fibroblasts. Increased miRNA from the Xq26.3 cluster were observed in both sample types. TGF-b upregulates these miRNA. Moreover, there is a strong inverse relationship between their levels and the predicted target in the TGF-b pathway (SMAD7). These miRNA are likely part of a positive feedback mechanism which is activated by TGB-b to target its natural inhibitor (SMAD7) keeping the pathway activated. Given the strong female predilection of SSc, this miRNA cluster, located on the X-chromosome, could have important ramifications for understanding SSc pathophysiology and drug development.
To cite this abstract in AMA style:Salazar G, Hagan J, Wu M, Guo X, Zhou X, Charles J, Mayes MD, Assassi S. The Global Microrna Profile of Systemic Sclerosis Whole Skin/Dermal Fibroblasts and the Role of the Xq26.3 miRNA Cluster As a TGF-b Pathway Positive Feedback Mechanism [abstract]. Arthritis Rheumatol. 2015; 67 (suppl 10). https://acrabstracts.org/abstract/the-global-microrna-profile-of-systemic-sclerosis-whole-skindermal-fibroblasts-and-the-role-of-the-xq26-3-mirna-cluster-as-a-tgf-b-pathway-positive-feedback-mechanism/. Accessed September 22, 2019.
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